Although the gustatory system provides animals with sensory cues important for

Although the gustatory system provides animals with sensory cues important for food choice and other critical behaviors little is known about neural circuitry immediately following gustatory sensory neurons (GSNs). the Syb-GRASP method suggested that the identified G2Ns receive synaptic inputs from are activated by sucrose trehalose and some other sugars (Thorne et Calpeptin al. 2004 Wang et al. 2004 Marella et al. 2006 Another set of Grs including is expressed in a different GSN population which responds to caffeine and other compounds that in high concentrations can sometimes be toxic (Thorne et al. 2004 Wang et al. 2004 Marella et al. 2006 Use of a genetic method led to the identification of a GSN population that participates in responses to low osmolality (Inoshita and Tanimura 2006 and later Nrp1 was identified as the gene responsible for taste detection in these GSNs (Cameron et al. 2010 Calpeptin is expressed in a different set of GSNs and responds to low concentrations (~100 mM) of sodium chloride (Zhang et al. 2013 Another population of GSNs is activated by carbonated water (Fischler et al. 2007 These subpopulations of GSNs distribute their axon terminals in distinct subregions in the PGC. evokes a series of feeding behaviors such as proboscis extension and cibarial pumping while activation of GSNs expressing antagonizes the activation of GSNs with (Marella et al. 2006 The neural circuits involved in these behaviors have been investigated. Proboscis extension is driven by a bilateral pair of motor neurons that elicits contraction of the muscles in the basal segment of the proboscis (Gordon and Scott 2009 and several additional neurons innervate the muscles of the cibarial pump (Manzo et al. 2012 A pair of cells identified as feeding command neurons induces a series of related behaviors: foreleg bending proboscis extension labellum opening and cibarial pumping (Flood et al. 2013 However these motor circuits do not receive direct synaptic input from the axon terminals of GSNs. In addition presentation of sugar or water to the proboscis functions as an unconditioned cue to reinforce appetitive learning about olfactory stimuli suggesting that taste information may be transmitted to the mushroom body a site of olfactory learning (Schwaerzel et al. 2003 Liu et al. 2012 Lin et al. 2014 Yet the neural pathways linking these regions have not been identified. Recently a group of Calpeptin projection neurons has been reported to be gustatory second-order neurons (Kain and Dahanukar 2015 These neurons identified through a behavioral screen and found to participate in proboscis extension make synaptic connections onto medium at 19-25°C. The following transgenic lines were used in this study: 1) (Gordon and Scott 2009 2 (Yoshihara and Ito 2000 Hayashi et al. 2002 3 (Jenett et al. 2012 4 (Jenett et al. 2012 5 (Jenett et al. 2012 6 (Jenett et al. 2012 7 (Verkhusha et al. 2001 8 (Gordon and Scott 2009 9 (Gordon and Scott 2009 10 (Lee and Luo 1999 12 (also known as (Wong et al. 2002 Calpeptin 14 (see below) 15 (Awasaki et al. 2011 16 (see below) 21 (Karuppudurai et al. 2014 22 (Wang et al. 2004 23 (Scott et al. 2001 24 (Zhang et al. 2013 25 (Cameron et al. 2010 26 (Zhang et al. 2013 27 (Potter et al. 2010 28 (Lai and Lee 2006 29 13 (attP2) (Karuppudurai et al. 2014 30 (Karuppudurai et al. 2014 To label single cells by the FLP-out recombination method wandering larvae or early pupae were treated with 39°C heat shock for 20 min (for fly strain first the 1469-base DNA fragment of the accompanied by signal peptide at its N-terminal was synthesized by a commercial service (Genscript Piscataway NJ): the sequence of the transgene was optimized for the codon usage of and sites were added at the 5’- and 3’-terminals respectively and the synthesized DNA was subcloned in pUC57. Second the fragment was subcloned into the pJFRC7-derived vector (Pfeiffer et al. 2010 which were also digested by the same set of enzymes. Third the resulting vector was digested by site on 3L. To generate the fly strain the 10.4 kb DNA enhancer fragment of R12C04 (Jenett et al. 2012 was amplified from the Canton-S genome by PCR (primer sequences 5′-CGGTATCTCAAGAATCGTCGCCATA-3′ and 5′-GCATGACCAATTCGTGTGGGTAAAC-3′) using Phusion High-Fidelity DNA polymerase (Thermo Scientific). The fragment was cloned using a pCR8/GW/TOPO TA Cloning Kit (Invitrogen) and was then transferred into a transformation vector (Pfeiffer et al. 2010 using Gateway LR Clonase II Enzyme mix (Invitrogen).The transgene was integrated into the site on 3L. The transgenes had been integrated using the typical PhiC31-mediated change process by Rainbow Transgenic Flies Inc. (Camarillo CA). The sequences of.